We have exciting new data showing reversal of new onset type 1 diabetes (T1D) (hyperglycemia, polyuria and weight loss) in nonobese diabetic (NOD) mice by treatment with an agonistic TLR4/MD-2 specific monoclonal antibody (UT18, hereafter referred to as TLR4-Ab). 90% of mice treated with TLR4-Ab showed a clinical response (delay in progression to endstage T1D) and 70% have permanent reversal of T1D. Successfully treated mice demonstrate decreased islet inflammation and preserved insulin staining of islet beta cells. Our approach is highly innovative because it targets a specific molecular complex, TLR4/MD-2, in the innate immune system to restore tolerance in the adaptive immune system. Although TLR4-Ab does not stimulate T cells directly, we show that immune tolerance can be restored to the adaptive immune system by this treatment: T cells from mice that had developed new onset diabetes, and then were successfully treated twice with TLR4-Ab, do not transfer disease. This approach has broad implications for T1D and other autoimmune diseases: the TLR4/MD-2 pathway is strongly evolutionarily conserved in humans compared to mice, providing a rationale to test this approach/immune pathway in humans. Moreover, there are already FDA approved drugs that target TLR4/MD-2 in humans, and more are actively being developed as a novel approach in cancer immunotherapy. We submit that this pathway is a promising new therapeutic approach for testing in autoimmunity. We propose to study the immunological mechanisms of reversal of T1D and restoration of adaptive immune tolerance achieved by this novel approach in two aims. In aim one, Defining the mechanisms of restored adaptive immune T cell tolerance in diabetic mice treated with TLR4-Ab, we will address the surprising finding that T cells isolated from mice that had developed new onset diabetes and were successfully treated with TLR4-Ab do not transfer diabetes. By what mechanism were these cells rendered unable to cause disease? Were CD4+ and CD8+ T effectors (Teffs) rendered tolerogenic or anergic, or were T regulatory cells (Tregs) increased in quantity or function - or were both Tregs and Teffs affected by treatment? We will perform studies with cell subtypes from antibody treated animals to identify whether Tregs and/or T effector cells are modulated by TLR4-Ab treatment. In Aim Two, Quantitative and functional analysis of the mechanistic effects of TLR4-Ab treatment on T effector and T regulatory cells in a T cell receptor transgenic transfer system, we will employ a rigorously defined T cell transgenic transfer system to quantify TLR4-Ab effects on specific cell subsets before, during and after onset of autoimmune diabetes, and map the fate and function of transgenic autoreactive T cells after TLR4-Ab treatment. Understanding the immunological mechanisms of disease reversal mediated by the TLR4 pathway is of tremendous interest and potential. These aims performed over two years should definitely establish the efficacy and mechanisms of anti-TLR4 treatment for future application to human T1D and other autoimmune diseases.